One of the best-documented effects of acute ethanol ingestion is the diuresis which follows within the first hour after drinking, related to athe drop in plasma vasopressin levels. During the past few years, we have used preparations of the intact posterior pituitary (neurophypophysis) as well as nerve terminals isolated from the pituitary, and have established that relevant concentrations of ethanol inhibit the release of vasopressin from the terminals, and that this inhibition of release results from the inhibition of voltage-gated calcium channels, and the potentiation of activity of calcium-activated potassium (CAK) channels in the terminal membrane. It is known that chronic ethanol exposure results in the development of tolerance to ethanol-induced diuresis. The wealth of information which we now possess regarding the acute actions of ethanol on the biophysics of release of vasopressin from the neurohypophysial terminals provides an ideal situation to study the molecular basis for the development of tolerance. In the proposed study, we will first carefully document the time course of development of tolerance to ethanol diuresis in rats. The neurohypophysis will then be removed from rats at various points in the tolerance cycle, and the characteristics of release induced by either elevated potassium or electrical stimulation will be obtained. Parallel experiments will test whether similar changes occur in isolated neurohypophyses chronically exposed to ethanol in organ culture, testing among other things, the role of transcription in the development of tolerance. Isolated terminals will be used from rats at various stages of the tolerance cycle, to examine release characteristics, and patch clamp experiments will determine the changes which occur in macroscopic and single channel properties of target channel populations during the development of tolerance. Finally, molecular biology techniques will be used to ascertain the changes in calcium and CAK channel subunit composition in the hypothalamic cell bodies of the terminals, which accompany development of tolerance to diuresis.